The overall goal of this research is to explore the biological function of protein methylation reactions, with particular attention to their possible roles in neuronal function. Our studies will focus on two distinct methyltransferase enzymes. One, protein L-isoaspartyl methyltransferase (PIMT). catalyzes methylation of atypical isoaspartyl sites (isoAsp) that arise in certain proteins as a form of spontaneous damage. IsoAsp can render proteins dysfunctional, and/or highly immunogenic in the same animal from which they are derived. Moreover, PIMT knock-out mice accumulate high levels of intracellular isoAsp sites and develop fatal epileptic seizures at 4-6 weeks. To understand more about the function of PIMT and the consequences of isoAsp accumulation, we will: (1) determine if PIMT functions in vivo to rescue damaged proteins or to facilitate their degradation by comparing the turnover rate of histone H2B (a major in vivo substrate for PIMT) in normal vs. PIMT-deficient cells, and by comparing the racemization of the isoAsp prone Asp-25 residue in H2B; (2) identify how isoAsp accumulation affects the function of synapsin-1 and other synaptosomal proteins; (3) determine if isoAsp sites greatly enhance the immunogenicity of mouse H2B in that same species, the extent to which isoAsp H2B induces an autoimmune pathology, and if the sera of patients afflicted with autoimmune diseases such as systemic lupus erythematosus have antibodies or T cells that selectively recognize the isoAsp form of H2B. The second enzyme to be studied is coactivator-associated arginine methyltransferase 1 (CARM 1), an enzyme that forms complexes with specific transcription factors that mediate glucocorticoid-regulated gene expression. To understand more about the function of CARM 1 we will (1) determine if purified HuD (an mRNA-binding protein implicated in neuronal development) is an in vivo substrate for CARM 1 and search for additional CARM 1 substrates in rat PC12 cells using a previously developed methyltransferase inhibitor-based strategy, and (2) determine if mammalian brain (or other tissues) contain a protein-arginine demethylating enzyme that may reverse the methylation reactions catalyzed by CARM 1 and/or related protein arginine methyltransferases. Our proposed studies on PIMT should provide new insights as to the possible contribution of isoAsp formation and PIMT deficiency in diseases afflicting the brain and possibly the immune system. Similarly, studies on CARM1 should provide important new information on how glucocorticoids regulate gene expression in the brain.